U.S. patent application number 15/454200 was filed with the patent office on 2017-09-14 for sensitive elisa for disease diagnosis on surface modified poly(methyl methacrylate) (pmma) microfluidic microplates.
The applicant listed for this patent is XiuJun Li, Sanjay S. Timilsina. Invention is credited to XiuJun Li, Sanjay S. Timilsina.
Application Number | 20170261504 15/454200 |
Document ID | / |
Family ID | 59786545 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170261504 |
Kind Code |
A1 |
Li; XiuJun ; et al. |
September 14, 2017 |
SENSITIVE ELISA FOR DISEASE DIAGNOSIS ON SURFACE MODIFIED
POLY(METHYL METHACRYLATE) (PMMA) MICROFLUIDIC MICROPLATES
Abstract
Certain embodiments are directed to an ultrasensitive
poly(methyl methacrylate) (PMMA) ELISA microfluidic microplate,
where the protein is covalently bound to a poly-lysine modified or
carboxylated PMMA surface.
Inventors: |
Li; XiuJun; (El Paso,
TX) ; Timilsina; Sanjay S.; (El Paso, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Li; XiuJun
Timilsina; Sanjay S. |
El Paso
El Paso |
TX
TX |
US
US |
|
|
Family ID: |
59786545 |
Appl. No.: |
15/454200 |
Filed: |
March 9, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62305622 |
Mar 9, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54353 20130101;
B01L 2300/0829 20130101; B01L 2300/0858 20130101; B01L 3/5085
20130101; G01N 33/54366 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; B01L 3/00 20060101 B01L003/00 |
Claims
1. An ultrasensitive microfluidic microtiter plate comprising a
plurality of microwells having a poly-lysine modified or
carboxylated poly(methyl methacrylate) (PMMA) capture surface,
wherein the plate has a limit of detection for IgG of 200
pg/mL.
2. The microfluidic plate of claim 1, further comprising a capture
protein covalently bound to the PMMA capture surface.
3. The microfluidic plate of claim 2, wherein the capture protein
is covalently bound through a poly-lysine modification or
carboxylation modification.
4. The microtiter plate of claim 2, wherein the capture protein is
an antibody or antibody fragment.
5. The microtiter plate of claim 1, wherein the plate comprises 8
or more microwells.
6. The microtiter plate of claim 5, wherein the plate comprises 96
to 800 microwells.
7. The microtiter plate of claim 5, wherein each microwell is about
0.001 to 3 mm in diameter and 0.01 to 4 mm in depth.
8. The microtiter plate of claim 1, wherein the microwells have a
flat or rounded floor.
9. The microtiter plate of claim 1, wherein the microwells are
funnel shaped, having an upper diameter of 0.1 to 5 mm and a lower
diameter of 0.001 to 1 mm.
10. The microtiter plate of claim 1, wherein the microwells are
arranged in an array.
11. The microtiter plate of claim 10, wherein the array is a radial
array or columnar array.
12. An enzyme linked immunosorbent assay (ELISA) kit comprising the
microtiter plate of claim 1.
13. The kit of claim 12, further comprising a capture agent.
14. The kit of claim 13, wherein the kit comprises a plurality of
capture agents.
15. The kit of claim 13, wherein the capture agent is coupled to
the microtiter plate.
16. The kit of claim 13, wherein the capture agent is provided in a
container separate from the microtiter plate.
17. The kit of claim 13, wherein the capture agent is an
antibody.
18. The kit of claim 13, further comprising detection reagents and
wash reagents.
19. A method for detecting an analyte in a sample comprising:
contacting an microtiter plate of claim 1, comprising a capture
agent covalently bound to the microtiter plate, wherein the capture
agent binds a target analyte, with a sample suspected of comprising
the target analyte; and detecting the presence of a signal.
20. The method of claim 19, wherein the signal is detected by
direct visualization or by or an office scanner.
Description
PRIORITY CLAIM
[0001] This application claims priority to U.S. Application No.
62/305,622 filed Mar. 9, 2016, which is incorporated herein by
reference in its entirety.
BACKGROUND
[0002] There is a fragile balance with respect to continual
emergence of new infectious disease and the reemergence of old
infectious disease, together with the potential for their global
spread (Nelson and Williams, Infectious disease epidemiology, Jones
& Bartlett Publishers, 2014). Infectious disease stand out
among many challenges to health because of their profound impact on
human species, unpredictable and explosive global effects due to
their pandemics. Unlike other diseases, which can result from
multiple interacting risk factors, most infectious disease are
caused by single agent, the identification of which leads not only
the control measures but treatment measures as well (Fauci and
Morens, New England Journal of Medicine, 2012, 366:454-461). This
underlines the need of accurate surveillance and the development of
new strategies for fast, rapid, and sensitive detection of
infectious diseases for their control.
[0003] Enzyme Linked ImmunoSorbent Assay (ELISA) is one of the most
commonly and widely used laboratory methods in medical diagnostic,
food industry (food allergen), plant pathology, quality control,
toxicology, and research applications. ELISA utilizes antibodies
and color change to identify a substance. This biochemical assay
detects proteins both quantitatively and qualitatively based on
their binding to immobilized antibodies or antigens. Most ELISAs
are performed in 96-well plates and take several hours to complete
because of the hour-long incubation and blocking time, as well as
consume large volumes of precious samples and reagents, and must be
performed in lab, which is not suitable for point-of-care
detection. Highly complicated and specialized instruments have been
developed to automate the assay, including robotic pipetters, plate
washers, and optical colorimetric detectors.
[0004] The sensitivity of the bioassay performed on a microfluidic
device depends upon the total activity of the proteins or enzymes
attached to the surface of the microfluidics chip. The interaction
of the protein with the chip surface has a profound effect on
sensitivity and specificity of the immunoassay. The hydrophobic
physical adsorption of proteins onto the microfluidics surface may
reduce functional site or activity of protein by even more then 90%
(Ren et al. Solid State Phenomena, 2006, 111:13-18; Tabata and
Ikada, Advanced drug delivery reviews, 1998, 31:287-301) and may
result in strong non-specific binding (Toepke and Beebe, Lab Chip,
2006, 6:1484-1486). So, to increase the sensitivity and binding
efficiency of the microfluidic chip, an appropriate surface
modification is required. Many different kinds of binding between
protein and the polymer surface has been defined; passive
adsorption on polymer beads (Sato et al. Analytical Chemistry,
2001, 73:1213-18), lipid layer grafted on PDMS (Mao et al.,
Analytical chemistry, 2002, 74:379-85), and protein A bound to PDMS
surface (Dodge et al., Analytical chemistry, 2001, 73:3400-09).
[0005] Functionalization of PMMA with amine groups have been
reported but consists of too many steps and yielded a low amine
density (Bulmu et al., Chemical Engineering Journal, 1997,
65:71-76) or involves unstable intermediates and non-environment
friendly solvents (Henry et al., Analytical chemistry, 2000,
72:5331-37).
[0006] Brown et al. (Lab Chip. 2006, 6(1):66-73) characterized the
surface modification of PMMA microfluidic devices. PMMA surfaces,
which were modified using air plasma, acid catalyzed hydrolysis,
and aminolysis (using ethylenediamine), to determine if covalent
and or hydrogen bonds between modified PMMA substrate and cover
plate increase the adhesion. Llopis et al. (Electrophoresis. 2007,
28(6):984-93) studied the surface modification of PMMA microfluidic
devices for high-resolution separation of single-stranded DNA. They
created an amine terminated PMMA surface by chemical or
photochemical process by using ethylenediamine, which was then used
to covalently anchor methacrylic acid used as scaffold to produce
linear polyacrylamides (LPAs). It helped them increase the
efficiency of separation of single stranded DNA
electrophoretically. Bai et al. (Langmuir. 2006, 22(22):9458-67)
studied the surface modification of PMMA to enhance the antibody
binding on polymer-based microfluidic device to perform ELISA. They
found that the poly(ethyleneimine) modified PMMA was 10 times more
active in binding antibodies as compared to those without treatment
or treated with small amine-bearing molecule, hexamethylenediamine
(HMD). They performed the ELISA of IgG with a similar detection
limit as the conventional 96-well plate microtiter plate. Bai et
al. (Biotechnol Bioeng. 2007, 98(2):328-39) performed ELISA of
Escherichia coli on PEI modified PMMA.
SUMMARY
[0007] Unspecific absorption of protein often leads to high
background and low sensitivity in enzyme linked immunosorbent assay
(ELISA). Covalent binding of proteins can enhance the binding
efficiency and improve the immunoassay sensitivity. Herein, the
inventors have developed a simple, miniaturized poly(methyl
methacrylate) (PMMA) ELISA microfluidic microplate, where the
protein is covalently bound to poly-lysine modified or carboxylated
PMMA surface. The modification with poly-lysine can be used to
aminate the PMMA surface. In certain aspects the carboxylated PMMA
surface is further modified to an amine-reactive sulfo-NHS ester.
In certain aspects the modified surface of PMMA is used for
covalently coupling peptides or polypeptide to the PMMA surface.
Unlike ELISA in traditional microplates, which is often limited by
long incubation and blocking time--rapid and ultrasensitive
detection of disease biomarkers can be completed within 90 min in
this microplate with much less reagent consumption. Immunoassays do
not require expensive and sophisticated equipment and results can
even be observed by the naked eye. Quantitative analysis can be
achieved by calculating the brightness of images scanned by a
desktop scanner. Although no specialized ELISA equipment was used,
the limits of detection of 200 pg/mL for Immunoglobulin G (IgG),
180 pg/mL for Hepatitis B surface antigen (HBsAg), and 300 pg/mL
for Hepatitis B core antigen (HBcAg) have been achieved using a
poly-lysine modified PMMA microplate, which are at least 10 fold
more sensitive as compared to commercial ELISA kits. In addition,
limits of detection of 190 pg/mL for IgG, 360 pg/mL for HBsAg, and
380 pg/mL for HBcAg have been achieved using carboxylated PMMA
surface which are also at least 10 fold more sensitive as compared
to commercial ELISA kits.
[0008] In certain aspects the microplate can comprise 8, 96, 192,
384, 800 or more microwells or chambers, including all values and
ranges there between. A microwell can be 0.001, 0.01, 0.1, 0.5, 1,
2, or 3 mm in diameter, including all values there between and
0.01, 0.1, 1, 2, 3, or 4 mm in depth, including all values there
between. In a further aspect, the microwells can be arranged in an
array. In certain aspects the array is, but need not be, a regular
array such as a linear or radial array. In certain aspects the
microwell array can be arranged in a 1, 2, 4, 6, 8, 10, 12 or more
rows by 1, 2, 4, 6, 8, 10, 12, 24 or more columns. In a further
aspect the array can be arranged in 2, 4, 5, 8, 10, 12 or more
radii. A horizontal cross section of the microwell can form any
geometric shape, such as a circular, square, rectangle, triangle,
etc. In certain aspects the microwell has a circular horizontal
cross section. In certain aspects the microwell can have a flat or
rounded floor. In certain aspects the detection well is a funnel
shaped well, with different upper well diameters (e.g. about 0.01,
0.1, 1, 2, 3, 4, or 5 mm, including all values and ranges there
between) and lower well diameters (e.g. 0.01, 0.1, 0.2, 0.3, 0.4,
0.5, to 1 mm, including all values and ranges there between). In
certain aspects the lower well diameters are smaller than upper
well diameters.
[0009] Certain embodiments are directed to methods of detecting an
analyte(s) comprising introducing a sample suspected of having or
comprising a target analyte(s) into a device described herein.
Subjecting the sample to detection or manipulation and detection,
wherein if a target is present in the sample an analyte binds to a
probe and produces a detectable signal. In certain aspects the
device is configured to detect a plurality of targets at once
(multiplexed assay) with a separate and distinct probe in an
individual detection microwell, or separate and distinguishable
probes in the same microwell.
[0010] The term "analyte" or "target analyte" refers to a compound
or composition to be detected or measured in the test sample. The
analyte will bind a probe, aptamer, or other capture agent. In
certain aspects the capture agent is covalently attached to a
surface. An analyte can be an antigenic substance, hapten, antibody
and combination thereof. The analyte of interest in an assay can
be, for example, a protein, a peptide, an amino acid, a nucleic
acid, a hormone, a steroid, a vitamin, a pathogenic microorganism,
a natural or synthetic chemical substance, a contaminant, a drug,
or metabolite.
[0011] The term "probe" or "capture agent" refers to a molecule
that can detectably distinguish between target molecules differing
in structure. Detection can be accomplished based on identification
of specific binding with a target. Examples of such specific
binding include peptides, proteins, antibodies, antibody fragments,
or other affinity reagents.
[0012] The term "antibody" as used herein includes immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen-binding site
that specifically binds (immunoreacts with) an antigen. The term
"antibody" as used herein also includes antibody-like molecules,
such as aptamers. A naturally occurring antibody (e.g., IgG, IgM,
IgD) includes four polypeptide chains, two heavy (H) chains and two
light (L) chains interconnected by disulfide bonds. However, it has
been shown that, fragments of a naturally occurring antibody can
perform the antigen-binding function of an antibody. Specific,
non-limiting examples of binding fragments encompassed within the
term antibody include (i) a Fab fragment consisting of the V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; (ii) an F.sub.d fragment
consisting of the V.sub.H and C.sub.H1 domains; (iii) an FIT
fragment consisting of the V.sub.L and V.sub.H domains of a single
arm of an antibody, (iv) a dAb fragment (Ward et al., Nature
341:544-546, 1989); and (vi) a F(ab').sub.2 fragment.
Immunoglobulins and certain variants thereof are known and many
have been prepared in recombinant cell culture (e.g., see U.S. Pat.
Nos. 4,745,055 and 4,444,487; WO 88/03565; EP 256,654; EP 120,694;
EP 125,023; Falkner et al., Nature 298:286, 1982; Morrison, J.
Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239,
1984).
[0013] The phrase "specifically binds" to a target refers to a
binding reaction that is determinative of the presence of the
target in the presence of a heterogeneous population of other
biologics. Thus, under designated conditions, a specified molecule
binds preferentially to a particular target and does not bind in a
significant amount to other biologics present in the sample.
[0014] As used herein, the term "sample" or "test sample" generally
refers to a material suspected of containing one or more targets.
The test sample may be used directly as obtained from the source or
following a pretreatment to modify the character of the sample. The
test sample may be derived from any biological source, such as a
physiological fluid, including, blood, interstitial fluid, saliva,
ocular lens fluid, cerebral spinal fluid, sweat, urine, milk,
ascites fluid, mucous, synovial fluid, peritoneal fluid, vaginal
fluid, amniotic fluid or the like. The test sample may be
pretreated prior to use, such as preparing plasma from blood,
diluting viscous fluids, lysing microbes in the sample, and the
like. Methods of treatment may involve filtration, precipitation,
dilution, distillation, mixing, concentration, inactivation of
interfering components, lysing organisms and/or cells, and the
addition of reagents. Besides physiological fluids, other liquid
samples may be used such as water, food products, and the like for
the performance of environmental or food production assays. In
addition, a solid material suspected of containing the target may
be used as the test sample. In some instances it may be beneficial
to modify a solid test sample to form a liquid medium or to release
a target (e.g., a nucleic acid).
[0015] Other embodiments of the invention are discussed throughout
this application. Any embodiment discussed with respect to one
aspect of the invention applies to other aspects of the invention
as well and vice versa. Each embodiment described herein is
understood to be embodiments of the invention that are applicable
to all aspects of the invention. It is contemplated that any
embodiment discussed herein can be implemented with respect to any
method or composition of the invention, and vice versa.
Furthermore, compositions and kits of the invention can be used to
achieve methods of the invention.
[0016] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0017] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0018] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0019] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0020] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0021] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of the specification
embodiments presented herein.
[0022] FIG. 1. Photograph of the actual device with black
background.
[0023] FIG. 2A-2B. Schematic of the covalent modification of PMMA.
A. poly-lysine modification; B. Carboxylation.
[0024] FIG. 3A-3C. FTIR analysis of surface modified PMMA. A.
poly-lysine modification, method 1 (poly-lysine in Dimethyl
sulfoxide (DMSO)); B. poly-lysine modification, method 2
(poly-lysine in Sodium hydroxide (NaOH) treated PMMA); C.
Carboxylation.
[0025] FIG. 4A-4B. Fluorescence intensity of surface modified PMMA.
A. poly-lysine modification; B. Carboxylation.
[0026] FIG. 5A-5B. Detection of IgG on poly-lysine modified PMMA
(#1 method). (A) Scanned image of enzymatic converted substrate in
different columns of the chip with concentrations from left to
right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 .mu.g/mL,
10 .mu.g/mL, and 100 .mu.g/mL, respectively. (B) Sigmoidal curve of
the corrected brightness of IgG over a concentration range of
10.sup.2 pg/mL to 10.sup.8 pg/mL.
[0027] FIG. 6A-6B. Detection of IgG on poly-lysine modified PMMA
(#2 method). (A) Scanned image of enzymatic converted substrate in
different columns of the chip with concentrations from left to
right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 .mu.g/mL,
10 .mu.g/mL, and 100 .mu.g/mL, respectively. (B) Sigmoidal curve of
the corrected brightness of IgG over a concentration range of
10.sup.2 pg/mL to 10.sup.8 pg/mL.
[0028] FIG. 7A-7B. Detection of HBsAg on poly-lysine modified PMMA
(#1 method). (A) Scanned image of enzymatic converted substrate in
different columns of the chip with concentrations, from left to
right: blank, 0.34 ng/mL, 3.4 ng/mL, 34 ng/mL, 340 ng/mL, 3.4
.mu.g/mL, 34 .mu.g/mL, and 340 .mu.g/mL, respectively. (B)
Sigmoidal curve of the corrected brightness of ELISA of HBsAg over
a concentration range of 34.times.10.sup.1 pg/mL to
34.times.10.sup.7 pg/mL.
[0029] FIG. 8A-8B. Detection of HBsAg on poly-lysine modified PMMA
(#2 method). (A) Scanned image of enzymatic converted substrate in
different columns of the chip with concentrations, from left to
right: blank, 0.34 ng/mL, 3.4 ng/mL, 34 ng/mL, 340 ng/mL, 3.4
.mu.g/mL, 34 .mu.g/mL, and 340 .mu.g/mL, respectively. (B)
Sigmoidal curve of the corrected brightness of ELISA of HBsAg over
a concentration range of 34.times.10.sup.1 pg/mL to
34.times.10.sup.7 pg/mL.
[0030] FIG. 9A-9B. Detection of HBcAg on surface modified PMMA
(poly-lysine). (A) Scanned image of enzymatic converted substrate
in different columns of the chip with concentrations from left to
right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 .mu.g/mL,
and 10 .mu.g/mL, respectively. (B) Sigmoidal curve of the corrected
brightness of HBcAg over a concentration range of 10.sup.2 pg/mL to
10.sup.7 pg/mL.
[0031] FIG. 10A-10B. Detection of IgG on surface modified PMMA
(carboxylation). (A) Scanned image of enzymatic converted substrate
in different columns of the chip with concentrations from left to
right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 .mu.g/mL,
10 .mu.g/mL, and 100 .mu.g/mL, respectively. (B) Sigmoidal curve of
the corrected brightness of IgG over a concentration range of
10.sup.2 pg/mL to 10.sup.8 pg/mL.
[0032] FIG. 11A-11B. Detection of HBsAg on surface modified PMMA
(carboxylation). (A) Scanned image of enzymatic converted substrate
in different columns of the chip with concentrations, from left to
right: blank, 0.34 ng/mL, 3.4 ng/mL, 34 ng/mL, 340 ng/mL, 3.4
.mu.g/mL, 34 .mu.g/mL, and 340 .mu.g/mL, respectively. (B)
Sigmoidal curve of the corrected brightness of ELISA of HBsAg over
a concentration range of 34.times.10.sup.1 pg/mL to
34.times.10.sup.7 pg/mL.
[0033] FIG. 12A-12B. Detection of HBcAg on surface modified PMMA
(carboxylation). (A) Scanned image of enzymatic converted substrate
in different columns of the chip with concentrations from left to
right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 .mu.g/mL,
and 10 .mu.g/mL, respectively. (B) Sigmoidal curve of the corrected
brightness of HBcAg over a concentration range of 10.sup.2 pg/mL to
10.sup.7 pg/mL.
[0034] FIG. 13A-13B. Multiplex assay in surface modified PMMA
(poly-lysine). Scanned image of the enzyme-catalyzed substrate, (A)
and bar plot of corrected brightness of the scanned image (B) for
detection of HBsAg and HBcAg. From left to right: immobilized
probe, none (1), HBsAg (2) and (3), HBcAg (4) and (5), and
HBsAg+HBcAg (6), (7), and (8), respectively. Test: From left to
right, solution containing, anti-HBsAg and anti-HBcAg (1) and (6),
HBsAg (2), (4), and (7), and HBcAg (3), (5), and (8). "a" and "b"
shows that the data are significantly different from each other at
p=0.05.
[0035] FIG. 14A-14B. Multiplex assay in surface modified PMMA
(carboxylation). Scanned image of the enzyme-catalyzed substrate,
(A) and bar plot of corrected brightness of the scanned image (B)
for detection of HBsAg and HBcAg. From left to right: immobilized
probe, none (1), HBsAg (2) and (3), HBcAg (4) and (5), and
HBsAg+HBcAg (6), (7), and (8), respectively. Test: From left to
right, solution containing, anti-HBsAg and anti-HBcAg (1) and (6),
HBsAg (2), (4), and (7), and HBcAg (3), (5), and (8). "a" and "b"
shows that the data are significantly different from each other at
p=0.05.
[0036] FIG. 15A-15B. Anti-interference test for the detection of
HBsAg in surface modified PMMA (poly-lysine). Corrected brightness
of the scanned image of ELISA as measured by ImageJ (A) and scanned
image of the chip (B) for the detection of HBsAg. From left to
right: detection of 0 ng/mL of HBsAg in the solution containing 1
.mu.g/mL HBcAg (1), 100 ng/mL CEA (2), 250 .mu.g/mL BSA (3), and 10
ng/mL PSA (4), respectively and 200 ng/mL of HBsAg in 1 .mu.g/mL
HBcAg (5), 100 ng/mL CEA+10 ng/mL PSA (6), 250 .mu.g/mL BSA (7),
and PBS (8), respectively. "a" and "b" shows that the data are
significantly different from each other at p=0.05.
[0037] FIG. 16A-16B. Anti-interference test for the detection of
HBsAg in surface modified PMMA (carboxylation). Corrected
brightness of the scanned image of ELISA as measured by ImageJ (A)
and scanned image of the chip (B) for the detection of HBsAg. From
left to right: detection of 0 ng/mL of HBsAg in the solution
containing 1 .mu.g/mL HBcAg (1), 100 ng/mL CEA (2), 250 .mu.g/mL
BSA (3), and 10 ng/mL PSA (4), respectively and 200 ng/mL of HBsAg
in 1 .mu.g/mL HBcAg (5), 100 ng/mL CEA+10 ng/mL PSA (6), 250
.mu.g/mL BSA (7), and PBS (8), respectively. "a" and "b" shows that
the data are significantly different from each other at p=0.05.
DESCRIPTION
[0038] Infectious diseases, cancer, and other diseases are often
diagnosed by immunoassay. Enzyme Linked ImmunoSorbent Assay
(ELISA), one of the most commonly and widely used laboratory
immunoassay methods in medical diagnostic and research
applications, detects proteins based on their binding to
immobilized antibodies or antigens. Even though, most ELISAs today
are performed in 96-well plates and are well suited for
high-throughput assays, highly complicated and specialized
instruments have to be utilized to automate the assay, including
robotic pipetters, plate washers, and spectrophotometers. These
traditional quantitative immunoassay experiments take several hours
to complete because of the hour-long incubation. Likewise, other
critical issue is the consumption of large volumes of precious
samples and reagents, and must be performed in a laboratory
setting, which is not suitable for point-of-care detection. The
progress in the field of microfluidics and MEMS technology could
provide an ELISA system for real time detection, multiplexing, and
reducing sample usage, for economical and high throughput diagnosis
(Herrmann, (2006) Lab on a Chip, 6, 555-560).
[0039] In certain aspects a microtiter plate is made from poly
(methyl methacrylate) (PMMA). PMMA has various advantages over
other expensive substrates. It is more rigid and less fragile,
disposable, and easy to fabricate using techniques such as hot
embossing or CO.sub.2 laser ablation. Also, it does not require the
longer fabrication and incubation time required for PDMS. In
certain aspects PMMA is modified with polylysine or carboxylation
so there is covalent binding of protein to the PMMA surface,
increasing its specificity and sensitivity. Certain aspects are
directed to multiplex detection of different biomarkers, for
example Hepatitis B (Hepatitis B Surface antigen (HBsAg) and
Hepatitis B core Antigen (HBcAg)). A microfluidics chip is created
for sensitive and specific multiplex disease detection using
modified PMMA, which can be both used in remote setting without
laboratory facility, and also in developed countries with
sophisticated instruments like microplate reader.
[0040] Hepatitis B virus (HBV) is used as a representative
disease/biomarker for proof of concept studies. HBV infection is a
major cause of chronic hepatic damage and of hepatocellular
carcinomas worldwide (Lai et al., The Lancet, 2003, 362:2089-94).
HBsAg, a qualitative serological biomarker for a developing HBV
infection, can diagnose acute and chronic hepatitis B virus
(Rodella et al., Journal of clinical virology, 2006, 37:206-12;
Jaroszewicz et al., Journal of hepatology, 2010, 52:514-22; Ben
Slama et al., Gastroenterologie Clinique et Biologique, 2010,
34:S112-S118). Also, the titer of serum HBsAg indicates the level
of infection and severity of disease (Ben Slama et al.,
Gastroenterologie Clinique et Biologique, 2010, 34:S112-S118; Ganem
and Prince, New England Journal of Medicine, 2004,
350:1118-29).
[0041] In one embodiment a microfluidic chip is prepared from
poly-lysine modified or carboxylated PMMA, which is a less
expensive replacement of microplate that can be used to read data
by microtiter plate or scanner for low resource setting. It is
economical; does not require trained personnel or considerable
volumes of biological samples. Similarly, all the reagent delivery
and washing steps can be integrated into the device so that it does
not require robust method of reagent delivery into each well
manually. In one aspect a funnel shaped PMMA has been created by
laser ablation of PMMA. In certain embodiments the ELISA takes
place on the upper surface of funnel, which is poly-lysine modified
or carboxylated.
[0042] Certain aspects of the invention are directed to
immunoassays, immunoassay devices, and immunoassay kits for
detecting one or more target analyte. Immunoassays generally
involve contacting with a sample directly or indirectly with a
capture agent. In certain aspects the capture agent can be a
polypeptide. In other aspects the capture agent can be directly or
indirectly linked to a solid support, i.e., modified PMMA as
described herein. Specific binding of a capture agent with a target
analyte from the sample can then be detected. In certain aspects
the capture agent is an antibody and the antibody can be detected
by an antibody detecting polypeptide, e.g., a secondary antibody.
In certain aspects the capture agent is covalently linked to the
solid support, i.e., the poly-lysine modified or carboxylated PMMA
microtiter plate described herein.
[0043] In a particular aspect, an immunoassay may be carried in one
or more of the following steps: (i) a poly-lysine modified or
carboxylated PMMA support is coated with a capture polypeptide or
agent (e.g., an antibody), (ii) the support is washed and then
blocked with a blocking buffer, (iii) the support is washed and a
detection reagent (e.g., a secondary antibody) is added, (iv) the
support can be washed and the appropriate detection reagents or
methodology added or performed; and (v) the support is examined or
assessed for any detectable signal.
[0044] In certain aspects a label is capable of generating a
measurable signal when it is contacted with the appropriate
substrate or stimulus (e.g., light comprising an appropriate
wavelength of electromagnetic radiation). In some embodiments, the
detection reagent is conjugated to a label selected from
horseradish peroxidase (HRP), I.sup.125, alkaline phosphatase,
fluorescein isothiocyanate (FITC), tetramethyl rhodamine
isothiocyanate (TRITC), green fluorescent protein (GFP),
allophycocyanin, phycocyanin, phycoerythrin, and
phycoerythrocyanin. In some embodiments, the detection reagent is
not labeled, and can be detected, for example, with a secondary
antibody, that is optionally labeled.
[0045] Other embodiments are directed to an assay kit comprising a
poly-lysine modified or carboxylated PMMA support. In one
particular aspect, the kit may contain an antibody capture
polypeptide, a capture antibody, and/or an antibody detection
polypeptide. The kits of the invention may further contain at least
one of following reagents for carrying out the immunoassay such as
blocking buffer, stopping reagents, a label substrate, and washing
solutions, for example.
[0046] According to other embodiments, an assay device used for
detecting target analytes may include a poly-lysine modified or
carboxylated PMMA support coated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more capture agents (e.g., capture polypeptide or an antibody).
The immunoassay device of the invention may include a capture agent
linked to the poly-lysine modified or carboxylated PMMA
substrate.
[0047] In certain aspects the poly-lysine modified or carboxylated
PMMA substrate can be part of a microfluidic chip. For example, the
chip used in this study was designed in Adobe Illustrator CS5 and
micro-machined using Laser cutter (Epilog Zing 16, Golden, Colo.).
In certain aspects the wells in the PMMA layer are designed
according to the dimension of standard 384 well plate. In one
example, as seen from FIG. 1, the X-axis and Y-axis offset is 4.5
mm (d), which, is the length between the centers of 2 wells. The
position of the first well corresponds to A1 of microtiter plate,
with the center of first well. A1 row offset of the chip is 8.99 mm
(a), similar to microtiter plate. In addition, A1 column offset of
12.13 mm (b) is same as microtiter plate. The diameter of each well
is 3.6 mm.
Covalent Modification of PMMA
[0048] FIG. 2A illustrates one method for covalent modification of
PMMA. First PMMA is sonicated for 10 minutes in 50% aqueous
2-propanol solution. PMMA is dried. After drying the dried PMMA is
immersed in poly-lysine (e.g., 0.2%, 0.1%, or 0.05% poly-lysine
solutions) in DMSO for 20 minutes at room temperature. PMMA is then
rinsed with 2-Propanol. Finally, PMMA is immersed in Glutaraldehyde
solution (1% w/v) at room temperature for 30 minutes. Once the PMMA
is poly-lysine modified a protein is added so that there is
covalent binding of protein to the PMMA surface.
[0049] In an alternatively method, PMMA is immersed in 1N NaOH
solution at 55.degree. C. for 30 minutes. Then PMMA is immerged in
a poly-lysine solution (e.g., 0.2%, 0.1%, or 0.05%) at pH 7 at room
temperature for 1 hour. Finally, PMMA is immersed in Glutaraldehyde
solution (1% w/v) at room temperature for 30 minutes. Once the PMMA
is poly-lysine modified, protein is added so that there is covalent
binding of protein to the PMMA surface.
[0050] In yet another method of modification PMMA can be
carboxylated (see FIG. 2B). Carboxylation methods include
sonication of PMMA for 10 minutes in 50% aqueous 2-propanol
solution. PMMA is dried. PMMA is then submerged in 3M sulfuric acid
at 60 degrees for 20 minutes. After that it is rinsed first with
water, then with 2-propanol and dried. Then EDC/NHS solution (0.35M
EDC+0.1M NHS) is added and incubated for 15-20 minutes. Once the
PMMA is modified, protein is added so that there is covalent
binding of protein to the PMMA surface.
FTIR Analysis of Modified PMMA Surface
[0051] From FT-IR spectrum of the poly-lysine modified PMMA, it can
be seen that there is a distinct absorption band from 1,150
cm.sup.-1 to 1,250 cm.sup.-1, which can be attributed to the
C--O--C stretching vibration. The two bands at 1,387 cm.sup.-1 and
750 cm.sup.-1 can be attributed to the .alpha.-methyl group
vibrations. The band at 986 cm.sup.-1 is the characteristic
absorption vibration of PMMA, together with the bands at 1,063
cm.sup.-1 and 841 cm.sup.-1. The band at 1,723 cm.sup.-1 shows the
presence of the acrylate carboxyl group. The band at 1,435
cm.sup.-1 can be attributed to the bending vibration of the C--H
bonds of the --CH.sub.3 group. The two bands at 2,995 cm.sup.-1 and
2,951 cm.sup.-1 can be assigned to the C--H bond stretching
vibrations of the --CH.sub.3 and --CH.sub.2-- groups,
respectively.
[0052] Poly-lysine modification method 1 (FIG. 3A). Some major
changes can be observed in the spectrum of PMMA once it was
modified. The presence of Amide I (1652 cm.sup.-1), Amide II (1533
cm.sup.-1) and C--N stretch after the treatment of PMMA with
polylysine shows that the PMMA is aminated by Polylysine. The
aminated PMMA was further treated with glutaraldehyde as can be
seen from C.dbd.O (1637 cm.sup.-1) and C--N (1442 cm.sup.-1)
stretch vibration to covalently bind the antibody to the PMMA
surface. The inventors see strong absorption for Amide I (1646
cm.sup.-1) and Amide II (1555 cm.sup.-1) after the addition of an
antibody, which proves the covalent binding of the antibody to the
modified PMMA surface.
[0053] Poly-lysine modification method 2 (FIG. 3B). Some major
changes can be observed in the spectrum of PMMA once it was treated
with NaOH to get hydroxyl group followed by treatment with
polylysine to get aminated PMMA. The presence of Amide I (1617
cm.sup.-1), Amide II (1534 cm.sup.-1) and C--N (1435 cm.sup.-1)
stretch after the treatment of PMMA with polylysine shows that the
PMMA is aminated by Polylysine. Also, we can see CH.sub.2
stretching mode of vibration at 2933 cm.sup.-1 and V1 proton mode
band of peptide at 3239 cm.sup.-1. The aminated PMMA was further
treated with glutaraldehyde as can be seen from C.dbd.O (1637
cm.sup.-1) and C--N (1440 cm.sup.-1) stretch vibration to
covalently bind the antibody to the PMMA surface. The inventors can
see strong absorption for Amide I (1643 cm.sup.-1) and Amide II
(1541 cm.sup.-1) after the addition of an antibody, which proves
the covalent binding of the antibody to the modified PMMA
surface.
[0054] FT-IR of carboxylated PMMA (FIG. 3C). From FT-IR spectrum of
the PMMA, it can be seen that there is a distinct absorption band
from 1,150 cm.sup.-1 to 1,250 cm.sup.-1, which can be attributed to
the C--O--C stretching vibration. The two bands at 1,387 cm.sup.-1
and 750 cm.sup.-1 can be attributed to the .alpha.-methyl group
vibrations. The band at 986 cm.sup.-1 is the characteristic
absorption vibration of PMMA, together with the bands at 1,063
cm.sup.-1 and 841 cm.sup.-1. The band at 1,723 cm.sup.-1 shows the
presence of the acrylate carboxyl group. The band at 1,435
cm.sup.-1 can be attributed to the bending vibration of the C--H
bonds of the --CH.sub.3 group. The two bands at 2,995 cm.sup.-1 and
2,951 cm.sup.-1 can be assigned to the C--H bond stretching
vibrations of the --CH.sub.3 and --CH.sub.2-- groups,
respectively.
[0055] Some major changes can be observed in the spectrum of PMMA
once it was modified. Treatment of PMMA with H.sub.2SO.sub.4
creates a carboxyl functional group in the PMMA surface. The
splitted C.dbd.O stretch vibration for the acrylated (1724
cm.sup.-1) and non-acrylated (1699 cm.sup.-1) carboxyl group proves
the formation of carboxyl functional group at the surface of PMMA.
Then, the well-known EDC/NHS method was used to treat the
carboxylated PMMA to get the amine-reactive sulfo-NHS ester, which
can be proved by the S.dbd.O (1347 cm.sup.-1) assymetric stretch,
S--O (853 cm.sup.-1) stretch and S.dbd.O (1152 cm.sup.-1) symmetric
stretch. A strong absorption for Amide I (1558 cm.sup.-1) and Amide
II (1646 cm.sup.-1) can be seen after the addition of the antibody,
which proves the covalent binding of the antibody to the modified
PMMA surface.
[0056] Once the PMMA was modified, 20 .mu.g/mL of Cy-3 IgG was
added to the PMMA surface and incubated for 20 minutes and washed
with PBST for three times. Fluorescence intensity was measured
before and after washing with PBST (FIG. 4A-4B). The covalent
modification method with highest antibody binding will be used in
subsequent experiment for multiplex infectious diseases
detection.
[0057] Detection of IgG. For IgG detection assay, the primary
Antibody, IgG (0.1 ng/mL-100 .mu.g/mL in 10 mM, pH 8.0 PBS) was
pipetted to the chip. After the chip was incubated with primary
antibody for 20 minutes, the unreacted PMMA surface was blocked
with Bovine Serum Albumin (4.5% BSA w/v in PBS+0.05% Tween 20) for
another 20 minutes. After that, it was washed with washing buffer,
PBST (10 mM, pH 7.4 PBS+0.05% Tween 20). Following washing,
anti-rabbit IgG-Alkaline phosphatase (6 .mu.g/mL) was added. It was
then incubated for another 7 minutes. Then, the final wash was done
with washing buffer for three times. Finally, the substrate for the
alkaline phosphatase, i.e., BCIP/NBT (Nitroblue
tetrazolium+5-bromo, 4-chloro, 3-indoyl phosphate) was added. NBT
is used with the alkaline phosphatase substrate BCIP in western
blotting and immunohistological staining and immunoassay
procedures. These substrate systems produce an insoluble NBT
diformazan end product that is blue to purple in color and can be
observed visually. After 10 minutes, the PMMA was scanned with
scanner and the brightness value was measured by using the software
ImageJ.
[0058] The limit of detection (LOD) is defined as the concentration
value that generates a signal three standard deviation above the
blank value. The calibration curve of IgG on surface modified PMMA
(#1 method) was linear over the range of 10.sup.2 pg/mL to 10.sup.6
pg/mL with a regression curve of y=9.72 log (x)+98.95
(r.sup.2=0.98). The LOD of IgG on surface modified PMMA (#1 method)
was found to be 200 pg/mL. (FIG. 5A-5B)
[0059] The calibration curve of IgG on surface modified PMMA (#2
method) was linear over the range of 10.sup.2 pg/mL to 10.sup.7
pg/mL with a regression curve of y=12.26 log (x)+42.49
(r.sup.2=0.98). The LOD of IgG on surface modified PMMA (#2 method)
was found to be 140 pg/mL. (FIG. 6A-6B)
[0060] The limit of detection (LOD) is defined as the concentration
value that generates a signal three standard deviation above the
blank value. The calibration curve of IgG on surface modified PMMA
was linear over the range of 10.sup.2 pg/mL to 10.sup.8 pg/mL with
a regression curve of y=17.86 log (x)+9.57 (r.sup.2=0.98). The LOD
of IgG on surface modified PMMA was found to be 190 pg/mL. (FIG.
10A-10B)
[0061] HBsAg Detection assay. Different concentration of HBsAg
(0.34 ng/mL-340 .mu.g/mL in 10 mM, pH 8.0 PBS) was introduced to
the wells in the modified PMMA surface. After the PMMA was
incubated with antigen for 20 minutes, the unreacted PMMA surface
was blocked with Bovine Serum Albumin (4.5% BSA w/v in PBS+0.05%
Tween 20) for another 20 minutes. After that, primary antibody
i.e., anti-HBsAg was added and incubated for 20 minutes. It was
washed once with washing buffer, PBST (10 mM, pH 7.4 PBS+0.05%
Tween 20). Following washing, alkaline phosphatase labelled
secondary antibody (6 .mu.g/mL) was added. It was again incubated
for another 7 minutes. Then, the final wash was done with washing
buffer for three times. Finally, the substrate for the alkaline
phosphatase, i.e., BCIP/NBT (Nitroblue tetrazolium+5-bromo,
4-chloro, 3-indoyl phosphate) was added. After 10 minutes, the PMMA
was scanned with scanner and the brightness value was measured
using the software ImageJ.
[0062] The calibration curve of HBsAg on poly-lysine modified PMMA
(#1 method) was linear over the range of 34.times.10.sup.1 pg/mL to
34.times.10.sup.6 pg/mL with a regression curve of y=13.13 log
(x)+75.1 (r.sup.2=0.98). The LOD of HBsAg on surface modified PMMA
(#1 method) was found to be 180 pg/mL. (FIG. 7A-7B)
[0063] The calibration curve of HBsAg on poly-lysine modified PMMA
(#2 method) was linear over the range of 34.times.10.sup.1 pg/mL to
34.times.10.sup.6 pg/mL with a regression curve of y=16.46 log
(x)+74.9 (r.sup.2=0.98). The LOD of HBsAg on surface modified PMMA
(#2 method) was found to be 160 pg/mL. (FIG. 8A-8B)
[0064] The calibration curve of HBsAg on carboxylated PMMA was
linear over the range of 34.times.10.sup.1 pg/mL to
34.times.10.sup.6 pg/mL with a regression curve of y=11.05 log
(x)+91.76 (r.sup.2=0.98). The LOD of HBsAg on surface modified PMMA
was found to be 360 pg/mL. (FIG. 11A-11B)
[0065] Detection of HBcAg. Different concentration of HBcAg (0.1
ng/mL-10 .mu.g/mL in 10 mM, pH 8.0 PBS) was introduced to the wells
in the modified PMMA surface. After the PMMA was incubated with
Antigen for 20 minutes, the unreacted PMMA surface was blocked with
Bovine Serum Albumin (4.5% BSA w/v in PBS+0.05% Tween 20) for
another 20 minutes. After that, primary antibody i.e., anti-HBsAg
was added and incubated for 20 minutes. It was washed once with
washing buffer, PBST (10 mM, pH 7.4 PBS+0.05% Tween 20). Following
washing, alkaline phosphatase labelled secondary antibody (6
.mu.g/mL) was added. It was again incubated for another 7 minutes.
Then, the final wash was done with washing buffer for three times.
Finally, the substrate for the alkaline phosphatase, i.e., BCIP/NBT
(Nitroblue tetrazolium+5-bromo, 4-chloro, 3-indoyl phosphate) was
added. After 10 minutes, the PMMA was scanned with scanner and the
brightness value was measured using the software ImageJ.
[0066] The calibration curve of HBcAg on surface modified PMMA was
linear over the range of 10.sup.2 pg/mL to 10.sup.7 pg/mL with a
regression curve of y=14.07 log (x)+29.64 (r.sup.2=0.98). The LOD
of HBcAg on surface modified PMMA was found to be 380 pg/mL. (FIG.
12A-12B)
[0067] Multiplex Detection. The surface modified PMMA was used for
simultaneous colorimetric detection of HBsAg and HBcAg. As shown in
the diagram first column is negative control without any antigen,
hence no color development. Second and third columns are for the
detection of HBsAg while fourth and fifth columns are for the
detection of HBcAg. Third and fourth columns do not develop color,
as they do not have the respective antibody against the antigen but
second and fifth columns develop color as they have their
respective antibody. Sixth, seventh and eight columns have both the
antigen i.e. HBsAg and HBcAg. All of them develop color as they
have their respective antibody or the mixture of both the antibody.
(FIG. 13A-13B and FIG. 14A-14B)
[0068] Anti-interference Test. The detection assay needs to have a
high anti-interference capability to screen various infectious
diseases as the serum contains complex ingredients consisting of
hundreds of different proteins with a wide range of concentration
that may interfere the detection of target proteins.
Anti-interference experiments were performed in the various columns
of the modified PMMA. The experiment shows the detection of HBsAg
200 ng/mL with and without various interfering proteins (1 .mu.g/mL
HBcAg, 100 ng/mL carcinoembryonic antigen (CEA), 250 .mu.g/mL BSA,
and 10 ng/mL prostate specific antigen (PSA)). As shown in the
diagram, first four columns do not contain HBsAg while the last
four columns contains 200 ng/mL of HBsAg with various concentration
of interfering proteins. In the absence of HBsAg, there is no
development of color. Furthermore, the color intensity for the
detection of 200 ng/mL of HBsAg in the presence of different
interfering protein was similar to the detection of 200 ng/mL of
HBsAg without the interfering protein. It demonstrates that even
1,250 times concentrated interfering proteins could not influence
the specific detection of HBsAg. (FIG. 15A-15B and FIG.
16A-16B).
* * * * *